Blood cells originate from a population of hematopoietic stem cells which are typically defined as pluripotent cells with self-renewal capacity. Although the regenerative capacity of the hematopoietic system is impressive, the replicative potential of stem cells nevertheless appears finite. Because the number of times stem cells can divide is an important consideration in the development of novel therapeutic strategies, including stem cell transplantation, ex vivo expansion and gene therapy, studies that may help define the replicative potential of stem cells are of general interest. Previous studies with purified human "candidate" stem cells have shown that their functional properties change dramatically during ontogeny, that loss in their proliferative potential correlates with measurable changes in the number of telomere repeats and that they express low levels of telomerase. Here we propose to study biological properties of normal and malignant hematopoietic stem cells in relation to telomerase expression and telomere length on individual chromosomes. For these studies, clonally propagated normal and leukemic precursor cells will be analyzed by novel quantitative fluorescence in situ hybridization techniques. The specific aims of the proposed studies are: 1) To analyze chromosome specific, replication-dependent telomere shortening in (sub-) clones of purified CD34+CD38- cells from fetal liver, cord blood and adult bone marrow; 2) to compare telomere karyograms of normal, myelodysplastic and leukemic cells from the same patient in order to study the role of telomeres in myelodysplastic disease and leukemic transformation; and 3) To study the expression of telomerase, telomerase mRNA and related factors in normal and malignant stem cell "candidates" in relation to their proliferative potential and examine the effect of telomerase inhibitors on chromosome specific telomere length and the proliferative properties of such cells. Taken together, these studies should clarify the role of telomeraes in the proliferation of normal and malignant hematopoietic stem cells and leukemia specific chromosomal abnormalities.